Gap junctions are pores that span the membranes of adjacent cells, and as such provide a means of electrical and cytoplasmic continuity between those cells. These trans-cellular channels are made up of joined hemichannels, each a hexamer of integral membrane protein subunits termed connexins (Cx). As a form of electrical connectivity between neurons, gap junctions have been implicated in the functioning of various neuronal networks including some within the crustacean stomatogastric ganglion, the vertebrate retina, the inferior olivary complex of mammals, and autonomic networks of the medulla oblongata, including the pre-B"tzinger complex (PBC). The principle investigator's laboratory is focused on clarifying neuronal network mechanisms that generate and pattern rhythmic bursts of neuronal population activity. The activity of such rhythmogenic networks is fundamental to essential functions ranging from walking to information processing. In the case of the PBC, such activity is related to the generation of inspiratory movements of breathing. Understanding the mechanisms of PBC function will ultimately be vital to understanding central respiratory disorders such as central hypoventilation syndrome, and Rett Syndrome. The findings a) that neurons within the rodent PBC express at least some Cx, possibly including Cx26, Cx32, and Cx36, and b) that putative gap junction blockers (uncouplers) affect the generation of bursting by the PBC suggest a role for gap junctions in PBC rhythmogenesis. However, in other neuronal populations, uncouplers exert non-specific effects on membrane properties. Moreover, the detection of certain Cx within the PBC has been questioned due to a potential lack of specificity by the method employed. Accordingly the issue of whether gap junctions as a form of intercellular communication are important to PBC function remains unresolved. Thus, the research proposed herein will further examine this issue by accomplishing two specific aims. The first aim is to determine the mechanisms by which putative gap junction uncouplers alter PBC output. To this end the cytoplasmic connectivity between PBC neurons, and the potential alteration of this continuity by an uncoupler (carbenoxolone, or CBX) and a control agent (glycyrrhizic acid or GZA), will be evaluated by a fluorescence recovery after photobleaching (FRAP) microscopy technique. Along with the FRAP study, the effects of CBX and GZA on membrane properties including input resistance and calcium currents will be examined. The second aim of this research is to further elucidate ontogenetic patterns of Cx expression within PBC neurons. This aim will be accomplished using in situ hybridization to detect transcripts for Cx26, Cx32, Cx36, Cx45, Cx47, and Cx59 in PBC neurons in tissue from mice from embryonic day 12 through postnatal day 21. Neuronal networks that even in isolation from sensory or descending inputs generate bursts of activity (i.e., central pattern generators) underlie various essential functions such as breathing and locomotion. Accordingly, studies examining the mechanisms of such rhythmogenic network function will provide a conceptual framework within which to examine disorders affecting repetitive behaviors. In relation to the research proposed herein, better understanding the mechanisms of pre-B"tzinger Complex function will ultimately be vital to understanding central respiratory disorders such as central hypoventilation syndrome, and Rett Syndrome. [unreadable] [unreadable]